5 - Heterobifunctional Cross-linkers

Abstract

Heterobifunctional reagents can be used to cross-link proteins and other molecules in a two- or three-step process that restricts the degree of polymerization often obtained using homobifunctional cross-linkers. Heterobifunctional conjugation reagents consist of two different reactive groups that can couple with two different functional targets on proteins and other macromolecules. The consequence is the ability to direct the cross-linking reaction to selected parts of target molecules, thus garnering improved control over the conjugation process. One protein is modified with a heterobifunctional reagent using the cross-linker's most reactive or most labile end. The modified protein is then purified from excess reagents by gel filtration or rapid dialysis. Most heterobifunctionals contain at least one reactive group that has extended stability in aqueous environments, thus permitting the purification of an activated intermediate before adding the second molecule to be conjugated. Heterobifunctional cross-linking reagents may also be used to site-direct a conjugation reaction toward particular parts of target molecules. Amines may be coupled on one molecule, while sulfhydryls or carbohydrates are targeted on another molecule. Directed coupling is often important in preserving critical epitopes or active sites within macromolecules. Heterobifunctional reagents containing one photoreactive end may be used to nonselectively insert into target molecules by UV irradiation. Ligands having specific affinity toward a receptor may be labeled with a photoreactive cross-linker, allowed to interact with its target, and then photolyzed to permanently label the receptor at its binding site. The photoreactive group is stable until exposed to high-intensity light at UV wavelengths. Photoaffinity labeling techniques are an important investigation tool for determining binding site characteristics. Cross-linkers may be selected based not only on their reactivities but also on the length and type of the cross-bridge they possess.